Capillary Occlusion Research Articles

Commentary on Some Recent Theses Relevant to Combating Aging: June 2020.

Theses reviewed in this issue include "Clearance of Capillary Occlusions Improves Cortical Blood Flow and Cognitive Function in Alzheimer's Mouse Models," "Dermato-Informatic Approaches to Understanding and Improving Lesional Diagnostic Expertise in Cutaneous Oncology," "Furthering the Scope, Understanding, and Application of Proteolysis Targeting Chimera," "Optimization and Application of Synthetic High-Density Lipoprotein (sHDL) System in Atherosclerosis and Glioma Therapy," "Regulatory T Cell Enriching Microparticles for Promoting Tolerance in Vascularized Composite Allotransplantation," and "Tau Prion Strains Induce Distinct Pathological Phenotypes In Vivo."

The changes of retinal capillary after anti-vascular endothelial growth factor therapy in patients with macular edema associated with retinal vein occlusion

At present, intravitreal injections of anti-VEGF agents is the main method for the treatment of macular edema secondary to retinal vein occlusion (RVO), which can significantly inhibit neovascularization, release macular edema and improve the vision of patients. However, VEGF is a survival factor of vascular endothelial cells, whether it can lead to the progress of retinal ischemia and it has an effect on retinal capillaries deserves our clinical attention. Most scholars currently think that the anti-VEGF agents will not aggravate the occlusion of retinal capillaries in the treatment of macular edema secondary to RVO from the aspects of the changes of perifoveal capillary arcade, the quantification of foveal avascular zone area, retinal nonperfusion area and retinal vascular density of the superficial and deep capillary plexus In addition, the changes of these indicators may be related to the number of times patients need treatment, visual prognosis and so on. In the future, with the gradual popularization of OCT angiography and the prolongation of the number and time of anti VEGF drug treatment, we look forward to the study of larger samples and longer follow-up time to further analyze the influence of the retinal capillary after anti-VEGF therapy in patients with macular edema associated with RVO. Key words: Retinal vein occlusion/complications; Macular edema/drug therapy; Angiogenesis inhibitors/therapeutic use; Capillaries; Review

The Oxygen Saturation in Vascular Abnormalities Depends on the Extent of Arteriovenous Shunting in Diabetic Retinopathy.

Diabetic retinopathy is characterized by disturbances in retinal blood flow mediated by capillary occlusion, intraretinal microvascular abnormalities (IRMAs), neovascularizations, and omega loops and reduplications. It is likely that the study of oxygen saturation in these abnormalities can provide knowledge about their role in the development of diabetic retinopathy. The oxygen saturation in IRMA vessels and venous loops and reduplications were studied in 40 diabetic patients with severe nonproliferative or proliferative diabetic retinopathy. The saturation values in the studied vascular abnormalities were compared to those of the larger retinal arterioles and venules. There was a similar oxygen saturation (mean ± SD) in IRMAs observed to connect arterioles with venules (78.6% ± 11.8%, n = 22) and IRMAs connecting venules with venules (79.2% ± 9.0%, n = 12; P > 0.999). The saturation in IRMAs was significantly lower (P < 0.0002) than in arterioles (97.4% ± 5.2%, n = 40) and significantly higher (P < 0.0001) than the saturation in omega loops and reduplications (54.2% ± 19.3%, n = 6), which in turn showed no significant difference from the saturation in the venules (61.8% ± 6.8%, n = 40, P = 0.4). The findings suggest that the oxygen saturation in vascular abnormalities in diabetic retinopathy depends on the extent of arteriovenous (A-V) shunting, with venous saturation due to no A-V shunting in venous loops and reduplications, and intermediate oxygen saturation due to moderate shunting in IRMAs. This may precede the development of neovascularizations with arterial oxygen saturation due to high A-V shunting.

Cilioretinal Artery Occlusion Associated With Optic Disc Edema

Purpose: This work investigates the clinical features of cilioretinal artery occlusion (CLRAO) in the setting of optic disc edema. Methods: A retrospective chart review of 3 patients with concomitant CLRAO and optic disc edema was performed. All patients underwent multimodal imaging and systemic workup. Results: The patients were female, had a median age of 32 years, and were otherwise healthy. Presenting visual acuities were hand motion, 20/1000, and 20/70. One patient had optic nerve enhancement on magnetic resonance imaging. Systemic workups were noncontributory. All patients had ischemic retinal whitening and inner-retinal thinning in the cilioretinal artery (CLRA) distribution. Fluorescein angiography showed no signs of capillary nonperfusion, vascular inflammation, or frank vein occlusion. One patient was lost to follow-up and 2 were treated with high-dose corticosteroids with improvement in visual acuity. Conclusions: CLRAO can be associated with optic disc edema in the absence of outright central retinal vein occlusion, giant-cell arteritis, or known systemic disease.

STAT3 activation in circulating myeloid-derived cells contributes to retinal microvascular dysfunction in diabetes

BackgroundLeukostasis is a key patho-physiological event responsible for capillary occlusion in diabetic retinopathy. Circulating monocytes are the main cell type entrapped in retinal vessels in diabetes. In this study, we investigated the role of the signal transducer and activator of transcription 3 (STAT3) pathway in diabetes-induced immune cell activation and its contribution to retinal microvascular degeneration.MethodsForty-one patients with type 1 diabetes (T1D) [mild non-proliferative diabetic retinopathy (mNPDR) (n = 13), active proliferative DR (aPDR) (n = 14), inactive PDR (iPDR) (n = 14)] and 13 age- and gender-matched healthy controls were recruited to the study. C57BL/6 J WT mice, SOCS3fl/fl and LysMCre/+SOCS3fl/fl mice were rendered diabetic by Streptozotocin injection. The expression of the phosphorylated human and mouse STAT3 (pSTAT3), mouse LFA-1, CD62L, CD11b and MHC-II in circulating immune cells was evaluated by flow cytometry. The expression of suppressor of cytokine signalling 3 (SOCS3) was examined by real-time RT-PCR. Mouse plasma levels of cytokines were measured by Cytometric Beads Array assay. Retinal leukostasis was examined following FITC-Concanavalin A perfusion and acellular capillary was examined following Isolectin B4 and Collagen IV staining.ResultsCompared to healthy controls, the expression of pSTAT3 in circulating leukocytes was statistically significantly higher in mNPDR but not aPDR and was negatively correlated with diabetes duration. The expression of pSTAT3 and its inhibitor SOCS3 was also significantly increased in leukocytes from diabetic mice. Diabetic mice had higher plasma levels of IL6 and CCL2 compared with control mice. LysMCre/+SOCS3fl/fl mice and SOCS3fl/fl mice developed comparative levels of diabetes, but leukocyte activation, retinal leukostasis and number of acellular capillaries were statistically significantly increased in LysMCre/+SOCS3fl/fl diabetic mice.ConclusionSTAT3 activation in circulating immune cells appears to contribute to retinal microvascular degeneration and may be involved in DR initiation in T1D.

Optical coherence tomography angiography in Purtscher-like retinopathy associated with dermatomyositis: a case report

PurposeTo describe a multimodal imaging diagnosis of retinopathy in dermatomyositis.Case presentationA 21-year-old white woman with a history of fatigue and a cutaneous rash complained of visual impairment in her left eye. A funduscopic examination showed multiple confluent cotton-wool spots in both eyes. Swept source-optical coherence tomography presented macular edema in both eyes; optical coherence tomography angiography revealed superficial and deep capillary occlusion in all areas affected by cotton-wool spots; and fluorescein angiography showed vascular walls enhancement, veins dilatation, and capillary leakage. After large doses of intravenously administered glucocorticoid therapy, followed by a cyclophosphamide regimen, best corrected visual acuity returned to 20/20 in both eyes.ConclusionsThis case report presents optical coherence tomography angiography clinical findings in a rare case of dermatomyositis-associated retinopathy, remarking the importance of a multi-imaging approach for a correct diagnosis and treatment of eye injuries, in order to avoid serious complications and permanent sequelae.

MON-224 DIAGNOSING RENAL THROMBOTIC MICROANGIOPATHY BASED ON HISTOPATHOLOGY IN RAPIDLY PROGRESSIVE RENAL FAILURE

Appart from glomerular and tubulointerstitial lesion of rapidly progressive renal failure (RPRF), renal vascular lesion is a critical prognostic finding in which renal thrombotic microangiopathy (TMA) present with severe clinical manifestation and have a high morality. The aim of this study were to describe (1) the histopatological features of TMA, (2) its classification and (3) the associated lesions in RPRF. We observed 133 kidney biopsies performed in patients presented with RPRF from 2013 to March 2018 by by using light microscopy. Renal TMA was classified as acute TMA, defined by luminal thrombi including fibrin and fragments of red blood cells and associated with ischemic capillaries, mesangiolysis and edematous and mucinous intimal thickening, often infiltrated with foam cells. Chronic TMA was characterized by onion-skin fibrointimal sclerosis. Chronic active TMA showed both acute and chronic changes. There were 38 cases of TMA diagnosed histopathologically including 31/38 (81.6%) lupus nephritis (LN) with 26/31 classIV, 1/31 class V, 1/31 class III-V, 2/31class IV-V; 6/38 (15.8%) IgA nephropathy (IgAN) and 1/38 (2.6%) focal segmental sclerosis (FSGS). All 45% acute TMA were LN. 23% chronic TMA included 13% LN, 8% IgAN and 2% FSGS. 32% Chronic active TMA were 24% LN and 8% IgAN. Only one LN (2.6%) showed thrombus in artery. 34.2% cases with capillary occlusion were LN. The associated arteriolar lesions including thrombi (55.3%), edematous and mucinous intimal thickening (73.7%), fibrointimal sclerosis (34.2%) and immunofluorescence staining of thrombi with fibrin (44.7%) presented both in LN and IgAN. 55% had ischemic glomeruli. The FSGS solely showed arteriolar fibrointimal sclerosis and ischemic loops suggested for chronic TMA. The mesangiolysis, endothelial swelling and subendothelial expansion were not recognised in our observation probably due to the limitation of the quality of the microscopic slides as well as the lack of electromicroscopic equipment. The duplication of the basement membrane features was not applied for defining chronic TMA in capillaries especially in LN because of its frequent presence in many chronic conditions. Pathological features of renal thrombotic microangiopathy can varies considerably and can be seen frequently in many different severe kidney conditions.

Morphophysiological characteristics of skin microcirculation types in patients with Dupuytren’s contracture

Aim– to investigate the patterns of cutaneous microcirculation and their relationship with structural vascular changes in palmar hypoderm in patients with Dupuytren’s disease.Material and methods. In 26 patients with Dupuytren’s contracture aged between 45 and 70 years, the microcirculation of palmar skin was assessed before the planned surgical treatment using ultrasound pulsed Doppler (Minimax-Doppler K, SP Minimax, St. Petersburg, Russia) with a high-frequency sensor of 20 MHz and laser Doppler flowmetry (BLF21, Transonic Systems Inc., USA). The local 3-minute arterial ischemic test was performed in all patients by putting the occlusion cuff on the forearm. Histological analysis of intra-operative tissue specimens was done using light microscopy (Carl Zeiss Primo Star microscope with 3.1 MP UCMOS video camera) MicroCapture Ver 6.6 program was used for data acquisition.Results. The normocirculatory type of hemodynamics (1) was found in 17 % of observations; hyperemic (2) – in 19 %, congestive-spastic (3) – in 42 %, and congestive-static (4) – in 21 %. Histologically type 1 was characterized with initial signs of constrictive arterial remodeling and capillary occlusion, 2 – with marked hyperemia of the microcirculatory bed and diapedesis of blood cells, inflammatory perivascular infiltrates, 3 – with significant narrowing and deformations of lumens in small arteries and hyalinosis of arterioles, 4 – with pronounced polymorphism of capillary loops, significant changes in arteries and veins.Discussion. Hyperemic type of microcirculation reflects high activity of autoimmune inflammation. Congestic-spastic type indicates a significant decrease in the reactivity of precapillary microvessels. Congestic-stasic type is accompanied by the most pronounced constrictive vascular remodeling and denervation of the vascular bed.Conclusion. Dupuytren’s contracture is characterized with predominance of pathological types of microcirculation in palmar skin, which must be taken into account in individualized protocols of additive therapy.

Oxyphor 2P: A High-Performance Probe for Deep-Tissue Longitudinal Oxygen Imaging

Quantitative imaging of oxygen distributions in tissue can provide invaluable information about metabolism in normal and diseased states. Two-photon phosphorescence lifetime microscopy (2PLM) has been developed to perform measurements of oxygen invivo with micron-scale resolution in 3D; however, the method's potential has not yet been fully realized due to the limitations of current phosphorescent probe technology. Here, we report a new sensor, Oxyphor 2P, that enables oxygen microscopy twice as deep (up to 600μm below the tissue surface) and with ∼60 times higher speed than previously possible. Oxyphor 2P allows longitudinal oxygen measurements without having to inject the probe directly into the imaged region. As proof of principle, we monitored oxygen dynamics for days following micro-stroke induced by occlusion of a single capillary in the mouse brain. Oxyphor 2P opens up new possibilities for studies of tissue metabolic states using 2PLM in a wide range of biomedical research areas.

ARTERIAL OXYGEN SATURATION IN NEOVASCULARIZATIONS IN PROLIFERATIVE DIABETIC RETINOPATHY.

Retinal neovascularizations in proliferative diabetic retinopathy have been proposed to develop from larger retinal venules. However, angiographic evidence suggests that the new vessels may originate from both arterioles and venules, and the vitreous oxygen tension near retinal neovascularizations is similar to that of retinal arterioles. An assessment of the oxygen saturation in neovascularizations may help characterizing the vascular origin of these vessels in proliferative diabetic retinopathy. Dual wavelength oximetry was used to study the oxygen saturation in arterioles, venules, and retinal neovascularizations in 40 eyes from 40 patients with proliferative diabetic retinopathy. The oxygen saturation was significantly lower in retinal venules than in arterioles and neovascularizations (P < 0.0001), and after a correction for the influence of vessel diameter, there was no significant difference between the oxygen saturation in retinal arterioles and neovascularizations (P = 0.71). Age at onset and duration of diabetes mellitus contributed significantly to the variation in oxygen saturation of the venules, whereas none of the clinical background parameters contributed to the variation in oxygen saturation in arterioles and neovascularizations. The oxygen saturation in retinal neovascularizations in proliferative diabetic retinopathy is similar to that of the arterioles. Neovascularizations may act as shunts to bypass areas of capillary occlusion.

Uric acid enhances alteplase-mediated thrombolysis as an antioxidant

Uric acid (UA) therapy may prevent early ischemic worsening after acute stroke in thrombolysis patients. The aim of this study was to examine the influence of UA on the thrombolytic efficacy of alteplase in human blood samples by measuring thrombolysis under flow conditions using a newly developed microchip-based flow-chamber assay. Human blood samples from healthy volunteers were exposed to UA, alteplase, or a combination of UA and alteplase. Whole blood and platelet-rich plasma were perfused over a collagen- and thromboplastin-coated microchip, and capillary occlusion was monitored with a video microscope and flow-pressure sensor. The area under the curve (extent of thrombogenesis or thrombolysis) at 30 minutes was 92% lower in the UA–alteplase-treated group compared with the alteplase-treated group. D-dimers were measured to evaluate these effects in human platelet-poor plasma samples. Although hydrogen peroxide significantly decreased the elevation of D-dimers by alteplase, UA significantly inhibited the effect of hydrogen peroxide. Meanwhile, rat models of thromboembolic cerebral ischemia were treated with either alteplase or UA–alteplase combination therapy. Compared with alteplase alone, the combination therapy reduced the infarct volume and inhibited haemorrhagic transformation. UA enhances alteplase-mediated thrombolysis, potentially by preventing oxidative stress, which inhibits fibrinolysis by alteplase in thrombi.

Vascular Nitric Oxide–Superoxide Balance and Thrombus Formation after Acute Exercise

An acute bout of strenuous exercise in humans results in transient impairment of nitric oxide (NO)-dependent function, but it remains unknown whether this phenomenon is associated with increased risk of thrombotic events after exercise. This study aimed to evaluate effects of a single bout of exhaustive running in mice on the balance of vascular NO/reactive oxygen species production, and on thrombogenicity. At different time points (0, 2, and 4 h) after exercise and in sedentary C57BL/6 mice, the production of NO and superoxide (O2) in aorta was measured by electron paramagnetic resonance spin trapping and by dihydroethidium/high-performance liquid chromatography-based method, respectively, whereas collagen-induced thrombus formation was analyzed in a microchip-based flow-chamber system (total thrombus-formation analysis system). We also measured pre- and postexercise plasma concentration of nitrite/nitrate and 6-keto-PGF1α. An acute bout of exhaustive running in mice resulted in decreased production of NO and increased production of O2 in aorta, with maximum changes 2 h after completion of exercise when compared with sedentary mice. However, platelet thrombus formation was not changed by exercise as evidenced by unaltered time to start of thrombus formation, capillary occlusion time, and total thrombogenicity (area under the flow pressure curve) as measured in a flow-chamber system. Strenuous exercise increased the plasma concentration of nitrite but did not affect nitrate and 6-keto-PGF1α concentrations. An acute bout of strenuous exercise in mice reduced NO and in parallel increased O2 production in aorta. This response was most pronounced 2 h after exercise. Surprisingly, the reduced NO and increased O2 production in mice after exercise did not result in increased platelet-dependent thrombogenicity. These results show that transient reduction in NO bioavailability does not modify thromboresistance in healthy mice after exercise.

Macular capillary recovery in systemic lupus erythematosus complicated by Kikuchi–Fujimoto disease

Few case reports have described vaso-occlusive retinopathy in systemic lupus erythematosus (SLE) using optical coherence tomography (OCT) angiography. Here we report the clinical features of a patient with SLE, complicated by Kikuchi-Fujimoto disease, who developed vaso-occlusive retinopathy. We then describe the subsequent recovery of the macular capillaries as assessed by OCT angiography. A 16-year-old male was referred to us with fever, a 1-month history of violaceous red papules and erythematous plaques on his face and a painful nodule in his right neck. We diagnosed him with SLE complicated by Kikuchi-Fujimoto disease through physiological assessment and histology from his neck lymph node and chin skin. Systemic steroids were prescribed as treatment. After remission, his fever and cervical lymph node swelling with pain recurred and he developed blurred inferior vision in his left eye. His best-corrected visual acuities were 1.0 and 0.1 in the right and left eyes, respectively. Extensive cotton wool spots were observed in the right fundus, and retinal capillary occlusions were detected by OCT angiography of the left eye. We diagnosed this case as vaso-occlusive retinopathy with SLE and increased immunosuppressive treatment together with anticoagulation therapy. Macular capillaries, observed by OCT angiography, gradually recovered function following assessment at 7 and 16months post-onset of the vaso-occlusive retinopathy. We reported a 1½-year course of vaso-occlusive retinopathy in a patient with SLE complicated by Kikuchi-Fujimoto disease. Occlusion of the retinal vasculature and the subsequent recovery of circulation are clearly observed by OCT angiography.

Edaravone, a Synthetic Free Radical Scavenger, Enhances Alteplase-Mediated Thrombolysis.

The combination of alteplase, a recombinant tissue plasminogen activator, and edaravone, an antioxidant, reportedly enhances recanalization after acute ischemic stroke. We examined the influence of edaravone on the thrombolytic efficacy of alteplase by measuring thrombolysis using a newly developed microchip-based flow-chamber assay. Rat models of embolic cerebral ischemia were treated with either alteplase or alteplase-edaravone combination therapy. The combination therapy significantly reduced the infarct volume and improved neurological deficits. Human blood samples from healthy volunteers were exposed to edaravone, alteplase, or a combination of alteplase and edaravone or hydrogen peroxide. Whole blood was perfused over a collagen- and thromboplastin-coated microchip; capillary occlusion was monitored with a video microscope and flow-pressure sensor. The area under the curve (extent of thrombogenesis or thrombolysis) at 30 minutes was 69.9% lower in the edaravone-alteplase- than alteplase-treated group. The thrombolytic effect of alteplase was significantly attenuated in the presence of hydrogen peroxide, suggesting that oxidative stress might hinder thrombolysis. D-dimers were measured to evaluate these effects in human platelet-poor plasma samples. Although hydrogen peroxide significantly decreased the elevation of D-dimers by alteplase, edaravone significantly inhibited the decrease. Edaravone enhances alteplase-mediated thrombolysis, likely by preventing oxidative stress, which inhibits fibrinolysis by alteplase in thrombi.

Photothrombotic Induction of Capillary Ischemia in the Mouse Cortex during in vivo Two-photon Imaging.

Photothrombosis of blood vessels refers to the activation of a circulating photosensitive dye with a green light to induce clotting in vivo (Watson et al., 1985). Previous studies have described how a focused green laser could be used to noninvasively occlude pial arterioles and venules at the brain surface (Schaffer et al., 2006; Nishimura et al., 2007; Shih et al., 2013). Here we show that small regions of the capillary bed can similarly be occluded to study the ischemic response within the capillary system of the mouse cerebral cortex. The advantage of this approach is that the ischemic zone is restricted to a diameter of approximately 150-250 μm. This permits higher quality two-photon imaging of degenerative processes that would be otherwise difficult to visualize with models of large-scale stroke, due to excessive photon scattering. A consequence of capillary occlusion is leakage of the blood-brain barrier (BBB). Here, through the use of two-photon imaging data sets, we show how to quantify capillary leakage by determining the spatial extent and localization of intravenous dye extravasation.

Dietary Compound Chrysin Inhibits Retinal Neovascularization with Abnormal Capillaries in db/db Mice.

Diabetic retinopathy (DR) develops in a significant proportion of patients with chronic diabetes, characterized by retinal macular edema and abnormal retinal vessel outgrowth leading to vision loss. Chrysin, a naturally-occurring flavonoid found in herb and honeycomb, has anti-inflammatory, antioxidant, and anti-cancer properties. This study sought to determine the protective effects of chrysin on retinal neovascularization with abnormal vessels and blood-retinal barrier (BRB) breakdown in 33 mM glucose-exposed human retinal endothelial cells and in db/db mouse eyes. High glucose caused retinal endothelial apoptotic injury, which was inhibited by submicromolar chrysin. This compound diminished the enhanced induction of HIF-1α, vascular endothelial growth factor (VEGF), and VEGF receptor-2 (VEGFR2) in high glucose-exposed retinal endothelial cells. Consistently, oral administration of 10 mg/kg chrysin reduced the induction of these proteins in db/db mouse eye tissues. In addition, chrysin restored the decrement of VE-cadherin and ZO-1 junction proteins and PECAM-1 in hyperglycemia-stimulated retinal endothelial cells and diabetic mouse retina, possibly maintaining tight cell-cell interactions of endothelial cells and pericytes. Anti-apoptotic chrysin reduced the up-regulation of Ang-1, Ang-2, and Tie-2 crucial to retinal capillary occlusion and BRB permeability. Furthermore, orally treating chrysin inhibited acellular capillary formation, neovascularization, and vascular leakage observed in diabetic retinas. These observations demonstrate, for the first time, that chrysin had a capability to encumber diabetes-associated retinal neovascularization with microvascular abnormalities and BRB breakdown.

Pericytes as Inducers of Rapid, Matrix Metalloproteinase-9-Dependent Capillary Damage during Ischemia.

Pericytes are a key component of the neurovascular unit and are essential for normal BBB function. However, during acute ischemia, we find that pericytes are involved in creating rapid and heterogeneous BBB disruption in the capillary bed. The mechanism by which pericytes contribute to BBB damage warrants further investigation, as it may yield new therapeutic targets for acute stroke injury and other neurological diseases involving capillary flow impairment.

The Development of a Thin-Filmed Noninvasive Tissue Perfusion Sensor to Quantify Capillary Pressure Occlusion of Explanted Organs

A new thin-filmed perfusion sensor was developed using a heat flux gauge, thin-film thermocouple, and a heating element. This sensor, termed "CHFT+," is an enhancement of the previously established combined heat flux-temperature (CHFT) sensor technology predominately used to quantify the severity of burns [1]. The CHFT+ sensor was uniquely designed to measure tissue perfusion on explanted organs destined for transplantation, but could be functionalized and used in a wide variety of other biomedical applications. Exploiting the thin and semiflexible nature of the new CHFT+ sensor assembly, perfusion measurements can be made from the underside of the organ-providing a quantitative indirect measure of capillary pressure occlusion. Results from a live tissue test demonstrated, for the first time, the effects of pressure occlusion on an explanted porcine kidney. CHFT+ sensors were placed on top of and underneath 18 kidneys to measure and compare perfusion at perfusate temperatures of 5 and 20 °C. The data collected show a greater perfusion on the topside than the underside of the specimen for the length of the experiment. This indicates that the pressure occlusion is truly affecting the perfusion, and, thus, the overall preservation of explanted organs. Moreover, the results demonstrate the effect of preservation temperature on the tissue vasculature. Focusing on the topside perfusion only, the 20 °C perfusion was greater than the 5 °C perfusion, likely due to the vasoconstrictive response at the lower perfusion temperatures.

Effect of Heart Rate and Use of Beta Blockers on Mortality After Heart Transplantation

Heart transplantation (HT) recipients may have tachycardia secondary to cardiac denervation. As higher heart rate predicts worse outcomes in cardiovascular disease, we hypothesized that tachycardia and nonuse of β blockers are associated with increased mortality after HT. All patients who underwent HT at our institution from 1987 to 2010 were included. The association of heart rate 3months after HT and β-blocker use during follow-up to mortality was assessed using Kaplan-Meier and multivariate Cox proportional hazards regression analyses adjusting for clinically relevant baseline variables. From 1987 to 2010, there were 493 HT. After excluding 29 who died within 3months and 3 with follow-up <3months, 461 HT recipients (50±2years; 20% women) were included. Over a follow-up of 12±7years, selected important univariate predictors of post-HT mortality were older age, male gender, higher body mass index, ischemic cardiomyopathy, longer post-HT intensive care unit stay, and hospitalization and at 3months, increased mean pulmonary artery pressure, right atrial pressure and pulmonary capillary occlusion pressure, higher heart rate, and nonuse of β blockers during follow-up. In multivariate analysis, older ager, longer hospitalization, higher mean pulmonary artery pressure, higher heart rate at 3months (hazard ratio 1.02 per beat, 95% confidence interval 1.008 to 1.035, p= 0.02) and nonuse of β blockers (hazard ratio 1.43, 95% confidence interval 1.002 to 2.031, p <0.05) were associated with mortality. In conclusion, in a large single-center cohort of HT recipients, higher heart rate and nonuse of β blockers were independently associated with higher mortality.

Plasminogen activator inhibitor type 1 in platelets induces thrombogenicity by increasing thrombolysis resistance under shear stress in an in-vitro flow chamber model

Despite the proven benefits of thrombolytic therapy with tissue plasminogen activator (t-PA) for peripheral thromboembolism, perfusion failure frequently occurs, particularly in arterial circulation. We evaluated how the modification of fibrinolytic activity affects thrombus formation under flow and static conditions. t-PA-treated human whole-blood samples (n=6) were perfused over a microchip coated with collagen and tissue thromboplastin at different shear rates, and thrombus formation was quantified by measuring flow pressure changes. For comparison, rotational thromboelastometry (ROTEM) was used to evaluate fibrinolytic activity under static conditions. At a shear rate of 240s-1, t-PA (200-800IU/ml) concentration-dependently delayed capillary occlusion, whereas at 600s-1, capillary occlusion was significantly faster and t-PA had limited effects, even at a supra-pharmacological concentration (800IU/ml). In contrast, 200IU/ml t-PA efficiently prevented clot formation in the ROTEM assay. The combined treatment of blood with a specific PAI-1 inhibitor (PAI-039) moderately enhanced the efficacy of t-PA, but only under flow conditions. In addition, 1:1-diluted blood samples of PAI-1-deficient (-/-) mice showed a significant delay of capillary occlusion at 240s-1, compared with those from wild-type mice (1.55 fold; P<0.001). This delayed occlusion was reproduced in samples containing platelets from PAI-1-/- and plasma from wild type, but was not observed by the opposite combination of blood components. The present results suggest that the anti-thrombotic efficacy of t-PA is sensitive to arterial shear flow, and that PAI-1 secreted from activated platelets plays an essential role in thrombolytic resistance.